The role of an endothelial specific g-protein coupled receptor in thrombosis
L. Gottlob1, P. Dusart1, L. Fagerberg2, D. A. Trégouët3, P. Morange4, M. Germain3, M. Civelek5, T. Renne1,6, J. Odeberg1, L. Butler1 (1Stockholm, Sweden, 2Solna, Sweden, 3Paris, France, 4Marseille, France, 5Charlottesville, United States, 6Hamburg, Germany)
Time: 17:15 - 18:15
Objective: Venous thromboembolism (VTE) is the third most common cardiovascular disease and affects roughly 1 in 1000 individuals annually. The role of the endothelium in VTE is understudied. We recently identified an endothelial enriched orphan g-protein coupled receptor (GPCR) that is expressed throughout the adult human vasculature. In a genome wide association study of VTE patients ‘MARTHA’ we found an association between this gene locus and the development of VTE. Using expression quantitative trait loci analysis we found that this VTE associated SNP was linked to reduced transcription of the GPCR in human endothelial cells (EC).The objective of this study was to investigate the possible role of this endothelial enriched orphan GPCR in VTE. Specifically, how this GPCR is regulated and if it modulates the expression of EC proteins important in the regulation of coagulation, cell signalling and blood clotting.
Methods: We used siRNA to deplete the GPCR in human umbilical vein endothelial cells (HUVEC). Effects of the siRNA-mediated depletion on the expression of a panel of EC coagulation and inflammation related proteins were assessed by real-time pcr, protein immunoblotting and quantitative mass spectrometry. We used a chromogenic activity assay and calibrated automated thrombography (specifically adapted to incorporate EC), to study the effects of the GPCR depletion on tissue factor activity in EC and thrombin generation in plasma respectively.
Results: The siRNA-mediated depletion of the orphan GPCR in HUVEC resulted in a significant increase in tissue factor (F3) transcription. The induction of F3 expression by tumour necrosis factor treatment of HUVEC was also significantly amplified following depletion. A significant increase in tissue factor activity was also observed after siRNA depletion of the GPCR. Using a calibrated automated thrombogram assay we also observed increased thrombin generation in plasma following depletion, an effect that was abolished when an inhibitory antibody against F3 was added.
Conclusion: Our results suggest that this GPCR could play a role in VTE development though the regulation of tissue factor expression.